This project explores the molecular mechanism of active transport of neurotransmitters, a process which plays an important role in synaptic transmission, both in the termination of the signal (high affinity cytoplasmic transporters) and in the scavenging and storage of the neurotransmitter (subcellular storage organelles). We have identified and purified proteins involved: the sodium- and chloride-coupled gamma-amino- butyric acid (GABA) and the sodium and potassium-coupled l-glutamate transporters from rat brain as prototypes of the transport process located in the synaptic plasma membrane, and the biogenic amine-proton antiporter as a prototype of those from the membrane of the storage organelles. We shall continue their biochemical characterization, with the emphasis on structure-function relationships at the level of the proteins themselves and possible modulation by phosphorylation. Furthermore, we have cloned the gene encoding the GABA transporter, sequenced it and expressed it in oocytes. We intend to clone also the gene coding for the biogenic amine transporter. Genes encoding for related transporters in the same or other tissues and in other organisms will be identified and sequenced, and expressed using transient and stable expression systems. We shall localize domains of the transporters suspected to be crucial for the functioning of the transporters by various techniques, such as, for instance, the formation of heterologous hybrids with cDNA's of other transporters, including the transporter clones isolated from the existing ones by low- stringency hybridization. Important amino acids will also be identified by chemical means. We shall use the above information for the generation and characterization of site-directed mutants. The distribution of the transporters will be monitored using in situ-hybridization and immunocytochemistry. In addition to understanding basic principles of catalysis by membrane proteins, our studies will enable us to test levels of expression of the transport proteins in pathological states in which neurotransmitter levels are altered.